Elucidation of the basic mechanisms of ribosome catalyzed translation of mRNA is critical to understanding both normal and aberrant processes of gene expression, cellular growth, and cellular differentiation. The principal catalytic activity of ribosomes, peptidyl transferase, is integral to the large ribosomal subunit. For E. coli ribosomes, the presence of only 23S rRNA and five of the more than thirty 50S subunit proteins correlates with the reconstitution of peptidyl transferase activity in vitro. The structural interactions of these proteins with 23S rRNA will be probed by in situ rRNA cleavage. Initial focus will be on protein L2, the protein most frequently implicated (along with 23S rRNA) in peptidyl transferase function Cysteine residues will be introduced into or removed from the proteins by site-directed mutagenesis, and para- bromoacetamido-benzyl-EDTA will be employed to covalently tether an Fe.EDTA moiety to each protein at unique sites. Individually, modified proteins will be used to reconstitute 50S subunits. Cleavage reactions will be performed, and the sites of rRNA cleavage will be identified by primer extension analysis. These experiments have the potential to reveal essential structural interactions that contribute to the formation of the peptidyl transferase active site, and to expand our knowledge of the higher order structure of the 50S subunit.